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| Item Type: | Preprint |
|---|---|
| Title: | Damaging RBM20 E-rich domain variants are not rescued by gene replacement |
| Creators: |
Bai, Fang (Flora)  ORCID: https://orcid.org/0000-0003-0639-8104, Chua, Kaiser ORCID: https://orcid.org/0009-0009-1730-6149, Li, Daniel ORCID: https://orcid.org/0000-0003-1015-6298, Kirillova, Anna, Yadav, Sunil, Staudt, David ORCID: https://orcid.org/0000-0002-3399-9311, Yamamoto, Yuta ORCID: https://orcid.org/0000-0001-6129-9745, De Jong, Hannah N. ORCID: https://orcid.org/0000-0002-8699-9702, Müller, Linda H. ORCID: https://orcid.org/0000-0002-9250-0541, Fenzl, Kai ORCID: https://orcid.org/0000-0001-8097-7740, Rungaramsin, Non, Huang, Yong ORCID: https://orcid.org/0009-0000-6727-4960, Reuter, Chloe ORCID: https://orcid.org/0000-0002-0429-9922, Zhang, Steven, Krysov, Vikki ORCID: https://orcid.org/0009-0007-5026-0475, Njoroge, Joyce ORCID: https://orcid.org/0000-0003-4379-5328, Floyd, Brendan J. ORCID: https://orcid.org/0000-0003-3185-0174, Lakdawala, Neal K. ORCID: https://orcid.org/0000-0001-6458-5421, James, Cynthia A. ORCID: https://orcid.org/0000-0001-8040-4600, Cannie, Douglas ORCID: https://orcid.org/0000-0001-6298-922X, Elliott, Perry ORCID: https://orcid.org/0000-0003-3383-3984, Mestroni, Luisa ORCID: https://orcid.org/0000-0003-1116-2286, Owens, Anjali ORCID: https://orcid.org/0000-0002-9669-8495, Merlo, Marco ORCID: https://orcid.org/0000-0002-1022-3131, Krahn, Andrew ORCID: https://orcid.org/0000-0002-7982-1586, Mao, Chad, Fatkin, Diane ORCID: https://orcid.org/0000-0002-9010-9856, Vedantham, Vasanth ORCID: https://orcid.org/0000-0003-1032-700X, Chahal, Anwar ORCID: https://orcid.org/0000-0001-7825-8827, Helms, Adam, Wu, Joseph C. ORCID: https://orcid.org/0000-0002-6068-8041, MacRae, Calum ORCID: https://orcid.org/0000-0001-5181-2664, Gotthardt, Michael ORCID: https://orcid.org/0000-0003-1788-3172, Roden, Dan ORCID: https://orcid.org/0000-0002-6302-0389, Roth, Fritz ORCID: https://orcid.org/0000-0002-6628-649X, Ashley, Euan ORCID: https://orcid.org/0000-0001-9418-9577, Meder, Benjamin ORCID: https://orcid.org/0000-0003-0741-2633, Kroncke, Brett ORCID: https://orcid.org/0000-0001-6215-0803, Tabet, Daniel ORCID: https://orcid.org/0009-0006-5480-7733, Cote, Atina, Steinmetz, Lars M. ORCID: https://orcid.org/0000-0002-3962-2865, Glazer, Andrew ORCID: https://orcid.org/0000-0002-3938-4713, Mercola, Mark ORCID: https://orcid.org/0000-0002-1430-2013 and Parikh, Victoria N. ORCID: https://orcid.org/0000-0002-5138-5559
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| Abstract: | The promise of precision therapeutics in genetic cardiomyopathies relies on linking specific therapies to variant mechanisms. Missense variants in the cardiac splice regulator RBM20 cause a highly penetrant and arrhythmogenic dilated cardiomyopathy. Disease-causing variants in RBM20’s arginine-serine rich (RS) domain act via formation of toxic gain of function cytoplasmic granules, but this is not true for a small number of clinically adjudicated pathogenic variants in its glutamate(E)-rich domain. To better define the effects of E-rich domain variants, we developed a scalable screen based on induced pluripotent stem cell (iPSC) cardiomyocyte differentiation that identified several additional damaging variants. Several of these reduced RBM20 protein abundance and stability. We therefore hypothesized that, unlike RS domain variants, these E-rich variants might be rescued by RBM20 overexpression. To test this hypothesis, we generated induced pluripotent stem cells (iPSCs) from a patient with a pathogenic E-rich domain variant (p.E913K), and confirmed reduced RBM20 protein expression in these RBM20(+/p.E913K) cells after differentiation to iPSC-derived cardiomyocytes (iPSC-CM, vs. engineered isogenic RBM20(+/+)). These iPSC-CMs also displayed aberrant transcriptional splicing, reduced contractility, increased calcium-induced calcium release, and nuclear localization of RBM20 protein, often with more than the two expected RBM20-centric splice factories. AAV-based overexpression of RBM20 reversed some, but not all of the mis-splicing events identified in RBM20(+/p.E913K) iPSC-CMs, and did not improve their abnormal contractility, calcium handling or supernumerary RBM20 nuclear granules. In summary, our data indicate that pathogenic E-rich domain variants reduce RBM20 protein abundance, but that their mechanism is unlikely to be explained by haploinsufficiency alone. |
| Source: | bioRxiv |
| Publisher: | Cold Spring Harbor Laboratory Press |
| Article Number: | 2026.07.13.738343 |
| Date: | 20 July 2026 |
| Official Publication: | https://doi.org/10.64898/2026.07.13.738343 |
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